Ohtsuki Kenzo, Hirayama Kyoko, Kawakami Fumitaka, Kato Tomoki, Kawakami Hiroshi
Laboratory of Genetical Biochemistry and Signal Biology, Graduate School of Medical Sciences, Kitasato University, Sagamihara 228-8555, Japan.
Biochim Biophys Acta. 2007 Aug;1770(8):1219-29. doi: 10.1016/j.bbagen.2007.04.013. Epub 2007 May 22.
By means of successive gel filtration on a Superdex 30 pg column and Mono S column chromatography, a 5-kDa polypeptide (p5) was highly purified from the low molecular weight (LMW) fraction separated from the partially purified lactoferrin (bLF) fraction of bovine milk, and biochemically characterized as a phosphate acceptor for two protein kinases [cAMP-dependent protein kinase (PKA) and casein kinase 1delta (CK1delta)] in vitro. Purified p5 was identified as a fragment (N-terminal positions 24-51, 28 amino acid residues) cleaved from fibroblast growth factor-binding protein (FGF-BP, p37). Both purified p5 and synthetic p5 (sp5) were effectively phosphorylated by PKA, and also phosphorylated by CK1delta in the presence of two sulfated lipids [sulfatide or cholesterol-3-sulfate (CH-3S), SCS] in vitro. A novel phosphorylation site (RNRRGS) for CK1delta and a potent SCS-binding site (RNRR) on p5 were identified. The PKA-mediated phosphorylation of p5 was highly stimulated when incubated with either acidic FGF (aFGF) or bLF in vitro, but this phosphorylation was more sensitive to SCS than H-89 (a specific PKA inhibitor). Immunoprecipitate experiments revealed p5, but not the phosphorylated p5, to be directly bound to aFGF in vitro. These results show that (i) p5 has a high binding affinity with aFGF as well as bLF; (ii) the binding of SCS to p5 results in the selective inhibition of its phosphorylation by PKA; and (iii) SCS functions as an effective stimulator for the phosphorylation of p5 by CK1delta in vitro. In addition, p5 may play an important physiological role as a trafficking factor for the physiological interaction between aFGF group including endothelial cell growth factors and their binding proteins in vivo.
通过在Superdex 30 pg柱上连续进行凝胶过滤和Mono S柱色谱法，从牛乳部分纯化的乳铁蛋白（bLF）组分中分离出的低分子量（LMW）级分中高度纯化出一种5 kDa的多肽（p5），并在体外将其生化特性鉴定为两种蛋白激酶[cAMP依赖性蛋白激酶（PKA）和酪蛋白激酶1δ（CK1δ）]的磷酸受体。纯化的p5被鉴定为从成纤维细胞生长因子结合蛋白（FGF - BP，p37）切割而来的片段（N端位置24 - 51，28个氨基酸残基）。纯化的p5和合成的p5（sp5）在体外均能被PKA有效磷酸化，并且在两种硫酸化脂质[硫苷脂或胆固醇 - 3 - 硫酸盐（CH - 3S），SCS]存在下也能被CK1δ磷酸化。鉴定出了p5上CK1δ的一个新的磷酸化位点（RNRRGS）和一个有效的SCS结合位点（RNRR）。当在体外与酸性FGF（aFGF）或bLF一起孵育时，PKA介导的p5磷酸化受到高度刺激，但这种磷酸化对SCS比对H - 89（一种特异性PKA抑制剂）更敏感。免疫沉淀实验表明，在体外p5而非磷酸化的p5直接与aFGF结合。这些结果表明：（i）p5与aFGF以及bLF具有高结合亲和力；（ii）SCS与p5的结合导致其被PKA磷酸化的选择性抑制；（iii）在体外SCS作为CK1δ对p5磷酸化的有效刺激剂发挥作用。此外，p5可能作为体内包括内皮细胞生长因子在内的aFGF组与其结合蛋白之间生理相互作用的转运因子发挥重要的生理作用。
